Essential Role of Jun Family Transcription Factors in PU.1-Induced Leukemic Stem Cell Transformation.

Knockdown of the expression of the myeloid master regulator PU.1 leads to the development of an immature acute myeloid leukemia (AML) in mice. Recent reports suggest that functional inactivation of PU.1 might also play a role in human AML. However, the molecular mechanisms underlying PU.1-mediated malignant transformation are unknown. We examined leukemic PU.1 knockdown mice and found a 3-fold expansion of lin-, c-kit+, Sca1+ (KLS) hematopoietic stem cells (HSC) as compared to wildtype controls, which was not observed during the preleukemic phase. When we transplanted double-sorted leukemic KLS-HSC into NOD-SCID mice the recipients developed AML after 9–12 weeks indicating that the leukemic stem cells derive from the HSC compartment. This finding prompted us to examine the transcriptome of PU.1 knockdown preleukemic HSC to identify early transcriptional changes underlying their malignant transformation. After lineage-depletion and FACS sorting of preleukemic KLS-HSC we performed linear amplification of RNA by 2 cycles of RT-IVT and hybridized the cRNA with Affymetrix Mouse Genome 430 2.0 arrays. Principal component analysis as well as hierarchical cluster analysis clearly distinguished PU.1 knockdown and wildtype HSC. Several in-vitro targets of PU.1 such as c-Fes, BTK, TFEC, CSF2R, and Ebi3 were downregulated demonstrating that those are also affected in HSC in vivo. Differential expression of 16 genes was corroborated by qRT-PCR. Strikingly, several Jun family transcription factors including c-Jun and JunB were downregulated. Retroviral restoration of c-Jun expression in bone marrow cells of preleukemic mice rescued the PU.1-initiated myelomonocytic differentiation block in this early phase. To target cells in the leukemic stage we applied lentiviral vectors expressing c-Jun or JunB. While c-Jun did not affect leukemic proliferation, lentiviral restoration of JunB led to an 80% reduction of clonogenic growth and a loss of leukemic self-renewal capacity in serial replating assays. Expression analysis of 285 patients with AML confirmed the correlation between PU.1 and JunB downregulation and suggests its relevance in human disease. These results delineate a transcriptional pattern that precedes leukemic transformation in PU.1 knockdown HSC and demonstrate that downregulation of c-Jun and JunB contribute to the development of PU.1-induced AML by blocking differentiation (c-Jun) and increasing self-renewal (JunB). Therefore, examination of disturbed gene expression in preleukemic HSC can identify genes whose dysregulation is essential for leukemic stem cell function and are potential targets for therapeutic interventions.